Introduction

A series of experiments observing Fluorescence Recovery After Photobleaching (FRAP) microscopy were used to find the recovery rates in lipid bilayers formed with dilauroylphosphatidylserine (DLPS), dilauroylglycerophosphocholine (DLPC),

dioleoylglycerophosphocholine (DOPC), and a fluorescently labeled dimyrsitoylglycerophosphoethanolamine formed in a high ionic strength buffer solution containing HEPES, magnesium chloride with varying concentrations of CHAPS detergent. These experiments were

run with 0X, 1X, and 5X CHAPS detergent initially. In each of these samples, there was a presence of round domains sporadically located on the bilayer, and FRAP techniques were used on both the bilayer and the round domains in each sample. Research was also done in

the absence of the fluorescently labeled lipid, using the same procedures as before, but then laying down a 50μL layer of 100nM Cytochrome P450 Reductase (a membrane protein) labeled with AlexaFluor 555 in HEPES, sodium chloride, and calcium chloride. Again, using

0X, 1x, and 5X concentrations of CHAPS detergent, FRAP techniques were used to note the phase segregation of the protein in the different domains and to measure the varying diffusion coefficients. Further experiments were conducted with labeled 2C9 Cytochrome P450

to determine co-localization with Cytochrome P450 Reductase within the heterogeneous biomimetic lipid assembly.

The Distribution of Cytochrome P450 Reductase and Cytochrome 2C9 Into Different

Phase Domains of Planar Biomimetic Lipid Assembly

David J. Wright, Dr. James A. Brozik, Dr. Jeff Jones

Washington State University, Department of Chemistry, Pullman, WA 99164

wrightdj@whitman.edu

Goals

1. Observe diffusion coefficients of round domains using FRAP techniques 2. Observe diffusion coefficients of lipid bilayer using FRAP techniques. 3. Observe behavior of Cytochrome 2C9 using FRET microscopy

FRAP Microscopy Setup

FRAP

Special thanks to the National Science Foundation, Adam Barden, Elsa Silva-Lopez, Angela Rudolph, and Adam Goler

for their support.

• Cytochrome 2C9 colocalized on the

DLPS, DOPC, and DLPC lipid bilayer

FRAP Results

Cytochrome 2C9 FRET Sample

•Cytochrome 2C9 after being

rinsed 7 times with 50 μL of

5X HEPES and magnesium

chloride and 1X CHAPS

detergent on the same sample

as above

References Gillam, E. M.J.; Guo, Z.; Ueng, Y.F.; Yamazaki, H.; Cock, I.; Reilly, P. E. B.; Hooper, W.D.; and Guengerich, P. F. Expression of Cytochrome P450 3A5 in Escherichia coli: Effects of 5’ Modification, Purification, Spectral Characterization, Reconstitution Conditions, and Catalytic Activities. Archives of Biochemistry and Biophysics. 1995, 317, 374-384. Imaoka, S.; Imai, Y.; Shimada, T.; and Funae, Y. Role of Phospholipids in Reconstituted Cytochrome P450 3A Form and Mechanism of Their Activation of Catalytic Activity. Biochemistry. 1992, 31, 6063-6069. Imaoka, S.; Terano, Y.; Funae, Y. Constitutive Testosterone 6β- Hydroxylase in Rat Liver. Journal of Biochemistry. 1988, 104, 481-487. Shaw, P.M.; Hosea, N.A.; Thompson, D.V.; Lenius, J.M.; Guengerich, F.P. Reconstitution Premixes for Assays Using Purified Recombinant Human Cytochrome P450A, NADPH-Cytochrome P450 Reductase, and Cytochrome b5. 1997, 348, 107-115.

Acknowledgments